Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobility–mass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.One of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N- and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3–9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation—i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patch-clamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13–15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach.     

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.Neurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson’s disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1–4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein–coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin– and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G protein–independent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracellular loop 3 by a fusion protein, as the intracellular domain is otherwise too small to provide crystal contacts. The fusion protein approach has provided a wealth of valuable structural data on GPCRs, but as it renders the crystallized constructs signaling-inactive, the most important functionality—the activation of G proteins—cannot be confirmed for these structures. This leads inevitably to a degree of uncertainty regarding the physiological relevance of intracellular structural aspects, and it also impedes the elucidation of signaling mechanisms, as functional assays and structure determination cannot be performed with the same GPCR constructs.Crystallization in the absence of fusion proteins was so far mainly possible for rhodopsin (12), the A_2A adenosine receptor (A2AR) (13), and the β₁-adrenergic receptor (14). Together, they share a high stability, which is either given naturally (rhodopsin) or it is due to stabilizing mutations. High stability appeared to be crucial for crystallographic success, as it allowed the application of harsh short-chain detergents. These tend to form small micelles, which may explain why crystal contact formation can occur under these conditions despite the small extra- and intracellular domains of class A GPCRs.Besides the stability requirement and/or the necessity of fusion proteins, structural studies of GPCRs have also been complicated by the need of eukaryotic expression systems [e.g., Spodoptera frugiperda (Sf9) insect cells], as prokaryotes exhibit generally low functional expression levels of wild-type GPCRs. However, prokaryotes such as Escherichia coli offer several advantages compared with insect cells, including quick genetic modification strategies, growth to high cell densities, fast doubling times, inexpensive media, absence of glycosylation, and robust handling. Furthermore, E. coli is well suited for producing fully isotope-labeled proteins—a crucial requirement for many NMR studies, which are limited to date.To exploit these advantages, we recently developed a directed evolution method for high functional GPCR expression levels in E. coli (15). In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10⁸ different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16, 17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli. Selection pressure for high functional expression levels was applied by incubating the induced cells with fluorescently labeled neurotensin, which allowed enrichment of the best expressing cells by fluorescence-activated cell sorting (FACS). The outlined procedure was performed in cycles, leading to a gradual adaptation of the NTR1 population toward high functional expression levels, and additionally, it gave rise to an increase in thermostability for certain variants.In a second technology, termed CHESS (cellular high-throughput encapsulation, solubilization and screening), we adapted this concept to directly evolve NTR1 variants for high thermostability in short-chain detergent micelles—a property that is not only beneficial for structural studies but also for in vitro drug screening (18). The crucial development of CHESS was to surround, simultaneously, every E. coli cell by a semipermeable polysaccharide capsule. This allows us to solubilize the receptor mutants with harsh short-chain detergents, each mutant inside its own encapsulated cell, all at once and in the same test tube. Both the solubilized receptors and their encoding plasmids are maintained within the same capsules. Long-term incubation under these conditions followed by labeling of the encapsulated solubilized receptors with fluorescent neurotensin and rounds of FACS enrichment ensured a strong selection pressure and a gradual adaption of the NTR1 population toward high stability in harsh short-chain detergents (18).In this work, we present the crystal structures of three evolved NTR1 variants, which were either obtained by evolving high functional expression levels in E. coli or by directed evolution for stability in detergent micelles. In contrast to the majority of crystallized GPCRs, our NTR1 variants are devoid of bulky modifications at the cytoplasmic face and can thus remain signaling-active, which allows us to gain unique insights into the structure–function relationship of NTR1.     

Accounting for a mirror-image conformation as a subtle effect in protein folding

By using local (free-energy profiles along the amino acid sequence and ¹³C^α chemical shifts) and global (principal component) analyses to examine the molecular dynamics of protein-folding trajectories, generated with the coarse-grained united-residue force field, for the B domain of staphylococcal protein A, we are able to (i) provide the main reason for formation of the mirror-image conformation of this protein, namely, a slow formation of the second loop and part of the third helix (Asp29–Asn35), caused by the presence of multiple local conformational states in this portion of the protein; (ii) show that formation of the mirror-image topology is a subtle effect resulting from local interactions; (iii) provide a mechanism for how protein A overcomes the barrier between the metastable mirror-image state and the native state; and (iv) offer a plausible reason to explain why protein A does not remain in the metastable mirror-image state even though the mirror-image and native conformations are at least energetically compatible.To perform their functions in living organisms, most proteins must fold from unfolded polypeptides into their functional, unique 3D structures. Understanding protein-folding mechanisms is crucial because misfolded proteins can cause many diseases, including neurodegenerative diseases (1) such as Alzheimer’s, Parkinson, and Huntington diseases. From theoretical and conceptual points of view, it has been suggested that a native protein exists in a thermodynamically stable state with its surroundings (2) and that a study of free-energy landscapes (FELs) holds the key to understanding how proteins fold and function (3, 4).The native structures of some proteins contain a high degree of symmetry that, in addition to the native structure, allows the existence of another, energetically very close to the native conformation, a native-like “mirror-image” structure. One of the representatives of such symmetrical proteins is the 10- to 55-residue fragment of the B domain of staphylococcal protein A [Protein Data Bank (PDB) ID: 1BDD, a three-α-helix bundle] (5). Protein A has been the subject of extensive theoretical (6–18) and experimental (19–23) studies because of its small size, fast folding kinetics, and biological importance. However, the mirror-image topology has never been a subject for discussion except for the earlier work by Olszewski et al. (7) and recent work by Noel et al. (24). The reason for this might be that it has never been detected experimentally and it was observed only in some theoretical studies (7–9, 12, 13, 15, 17, 18, 24) with different force fields. It is of interest to determine how realistic the mirror-image conformation is. Is it an artifact of the simulations or is it a conformation difficult to observe experimentally? Noel et al. (24) showed that the native and mirror-image structures have a similar enthalpic stability and are thermodynamically competitive and that the mirror image can be considered not just a computational annoyance, but as a real conformation competing with the native structure. Moreover, the mirror-image conformation is more entropically favorable than the native conformation (24). By making multiple mutations in the hydrophobic core and the first loop region, Olszewski et al. (7) found that the change in the handedness of the first loop induced by the mutations, the burial of the N cap of the second helix, and repacking of the hydrophobic core are responsible for formation of the mirror-image conformation. However, at the end, the authors stated: “… Whether the conclusion about the possible importance of turns in defining the global topology holds in general or is just specific to the three-helix bundles analyzed here requires additional investigation....” (ref. 7, p. 298).The difficulties for experiments to detect the mirror-image topology arise because the secondary structures of the mirror-image and the native conformation are identical and the native-contact interactions are similar in both conformations (details in Fig. S1 and SI Native and Mirror-Image Structures of Protein A). Hence, with an experimental technique such as circular dichroism, used to estimate the fraction of secondary-structure content, it is almost impossible to distinguish the mirror-image structure from the native structure. It would have been desirable if the mirror-image conformation and its evolution to the native structure could be detected by NMR spectroscopy. Nevertheless, by using local [¹³C^α chemical shift (25) and free-energy profiles (FEPs) along the amino acid sequence (26–28)] and global [principal component (PC) (29)] analyses (SI Materials and Methods), we examined molecular dynamics (MD) trajectories of protein A, generated with the coarse-grained united-residue (UNRES) force field (27, 30–32) (Fig. S2 and SI Materials and Methods). These analyses of the MD trajectories, in which folding from a fully unfolded conformation occurs either almost instantly or through a metastable state formed by the mirror-image topology, enabled us to elucidate the origin of the formation of a mirror-image topology and how the protein emerges from the kinetic trap and folds to the native state.The results presented in this work are based on the analysis of four pairs of MD trajectories at 270 K (in each pair, one trajectory folds directly to the native state and the other folds through the metastable mirror-image state) selected from 96 MD simulations, which we carried out in a broad range of temperatures (details in Materials and Methods). The mirror-image conformation is energetically competitive with the native conformation in the studied trajectories (an illustrative example of two trajectories is in Fig. S3), and these results are in agreement with those of earlier studies (12, 24).     

急性冠状动脉综合征病人血清CRP与纤溶活性变化

目的探讨急性冠状动脉综合征（ACS）病人血清C-反应蛋白（CRP）与纤溶活性变化。方法检测并比较47例ACS病人、41例稳定型心绞痛（SA）病人和40例正常人（正常对照组）CRP、纤维蛋白原（FIB）、血脂、D-二聚体浓度，组织型纤溶酶原激活物（tPA）、纤溶酶原激活物抑制物-1（PAI-1）活性及自细胞（WBC）总数。结果与SA组及正常对照组比较，ACS组病人CRP、FIB、WBC、D-二聚体及PAI-1明显升高（F=6．027～2543．668，q=3．571～16．098，P〈0．05、0．01），tPA降低（F=4．138，q=3．043～3．913，P〈0．05）；CRP与D-二聚体及PAI-1活性呈正相关（r=0．326、0．393，P〈0．05、0．01），与tPA活性呈负相关（r=-0．387，P％0．05）。结论ACS病人炎症标志物水平增高及纤溶活性降低，CRP水平升高与纤溶活性降低密切相关。炎症反应及纤溶活性降低在ACS的发生发展中具有重要作用。     

热休克蛋白表达对缺血性脑损伤的保护作用

INTRODUCTIONThecytoprotectioneffectofcerebralischemicpreconditioning(CIPhasbeenprovedbymanyfacts,buttheconcretemechanismofitre-maintobesolved.Thelateststudiespointoutthatpreconditioningischemiamaygeneratetheinternalantitraumicprocessinbodycelandinducetheischemicpreconditioningadaptationthuseliminatethbrainischemictrauma犤1,2犦.Heatshockprotein(HSPs)istheseriesoemergentproteinresultingfromthebadstimulation,whichprotectthebodycellfrominjuries.HSPsarethoughttobethebasicmaterialfotheintern…     

结缔组织生长因子在大鼠肾间质纤维化中的作用及辛伐他汀对其的影响

目的:本实验主要研究羟甲基戊二酰辅酶A(HM G-COA)还原酶抑制剂(HR I)辛伐他汀(sim vastatin)对单侧输尿管梗阻大鼠(UUO)肾间质纤维化的影响及可能机制。方法:将26只大鼠随机分为3组:假手术组:6只;UUO组:10只;sim vastating干预组:10只。结扎单侧输尿管造成梗阻性肾病模型,术后9 d处死大鼠,取梗阻侧肾做组织学检查,观察肾间质纤维化的程度,用免疫组化方法检测肾组织中结缔组织生长因子(CTGF)、α-平滑肌肌动蛋白(-αSMA)、胶原-Ⅳ(COL-Ⅳ)的水平。结果:组织学检查发现模型组出现了明显的肾小管扩张,上皮细胞萎缩,间质炎性细胞浸润,间质纤维化,而给药组肾间质纤维化程度与模型组相比有所减轻。免疫组化结果半定量分析显示给药组CTGF(0.997 8±0.115 5)、-αSMA(0.5523±0.0631)、COL-Ⅳ(1.062 9±0.054 0)的水平较模型组明显减低(P<0.05)。结论:辛伐他汀可以抑制CTGF、抑制肾小管上皮细胞的转分化、减轻细胞外基质的积聚,从而减轻间质纤维化,发挥肾保护作用。     

腮腺癌中Hsp70、p27的表达及意义

目的研究热休克蛋白70(Hsp70)、细胞周期调节蛋白p27在腮腺癌中的表达,并探讨与腮腺癌发生、发展之间的关系及临床意义。方法采用免疫组化通用型二步法检测45例腮腺癌(24例腺样囊性癌及21例粘液表皮样癌)中Hsp70、p27的表达,并选用10例多形性腺瘤作对照。结果Hsp70及p27的表达均为细胞核及胞浆中棕黄色染色,在腮腺癌中的阳性表达分别为77·8%及28·9%,与对照组多形性腺瘤比较差异显著(P<0·01)。Hsp70在腮腺癌中的表达与临床分期、组织学分级呈正相关(P<0·05);p27在腮腺癌中的表达与临床分期、组织学分级呈负相关(P<0·05)。结论Hsp70与p27在腮腺癌中的表达呈负相关(P<0·05),Hsp70与p27可作为判断患者预后的指标。     

耐甲氧西林金黄色葡萄球菌PBP2a 382～443片段的原核表达及鉴定

目的对编码耐甲氧西林金黄色葡萄球菌(MRSA)及青霉素结合蛋白2a(PBP2a)382~443位氨基酸的mecA基因片段进行克隆、表达及鉴定。方法根据基因文库登录的mecA基因的编码序列,针对编码PBP2a382~443位氨基酸的mecA基因片段,设计合成4条寡核苷酸片段,再将4条片段人工拼接成目的基因片段,然后克隆至PET-His载体,经酶切鉴定、测序正确后,转化E.coliBL21(DE3)plysS,用IPTG进行诱导表达,并对表达的蛋白以MRSA胶乳凝集试剂盒进行鉴定。结果构建了相应的PET-His克隆,经诱导表达和鉴定,证实成功表达出目的蛋白。结论成功表达出PBP2a382~443片段,为其进一步的纯化和应用奠定了基础。     

Novel strategies for rapid identification and susceptibility testing of MRSA

ABSTRACT

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with adverse clinical outcomes and increased morbidity, mortality, length of hospital stay, and health-care costs. Rapid diagnosis of MRSA infections has been associated with positive impact on clinical outcomes.     

Hypofunctionality of Gi proteins as aetiopathogenic mechanism for migraine and cluster headache

The involvement of Gi proteins in the modulation of pain perception has been widely established, and mutations in G-proteins have already been identified as the aetiopathological cause of human diseases. The aim of the present study was to determine whether a deficiency or a hypofunctionality of the Gi proteins occurred in primary headache. The functionality and the level of expression of Gi proteins were investigated in lymphocytes from migraine without aura, migraine with aura and cluster headache sufferers. A reduced capability to inhibit forskolin-stimulated adenylyl cyclase activity in headache patients was observed. Migraine patients also showed basal adenosine cAMP levels about four times higher than controls. The reduced activity of Gi proteins seems not to be related to a reduction of protein levels since no significant reduction of the Gialpha subunits was observed. These results indicate Gi protein hypofunctionality as an aetiopathogenic mechanism in migraine and cluster headache.